C-type lectin-like receptor (CLEC)-2, the ligand of podoplanin, induces morphological changes in podocytes

Podoplanin (PDPN) is intensely expressed on the podocyte membrane in an evolutionally conserved manner. CLEC-2, the endogenous ligand of PDPN, is highly expressed in platelets and also exists in a soluble form in plasma. Normally, podocytes are sequestered from CLEC-2, but when the glomerular barrier is injured, podocytes gain access to CLEC-2. We tested the effects of CLEC-2 in podocytes in vitro and in vivo. Cultured podocytes treated with Fc-CLEC-2 demonstrated that CLEC-2 induced the dephosphorylation of ezrin, radixin, and moesin (ERM) proteins. Podocytes treated with Fc-CLEC-2 also showed the dissociation of F-actin filaments from PDPN, F-actin degradation, detachment, and round morphology. Next, we perfused normal mouse kidney in vivo with FLAG-CLEC-2. CLEC-2 induced dephosphorylation of ERM and widening of the foot processes of podocytes. Platelets were detected by immunostaining for CD41 in the urine of mice with podocyte injury, indicating that podocytes can encounter platelets when glomeruli are injured. Collectively, these observations suggest that when platelets leak through the injured glomeruli, CLEC-2 from the platelets acts on PDPN in podocytes and induces morphological change and detachment, which may further aggravate podocyte injury. Thus, PDPN on podocytes may work as a leaked-platelet sensor.

www.nature.com/scientificreports/ that PDPN is important for maintenance of the normal function of podocytes. The cytoplasmic tail of PDPN interacts with ezrin, radixin, and moesin (ERM) proteins, which bind the actin cytoskeleton and regulate cell shape. It was reported that whole-body Pdpn-gene-disrupted mice showed no abnormal renal phenotypes 19 .
Podocalyxin-NHERF2 complex and nephrin-ephrin-B1-NHERF2 complexes can bind and maintain ezrin-F-Actin complex 20,21 . This may be a reason for the lack of abnormal phenotype in podocytes of congenital Pdpndeficient mice. However, suppression of PDPN by siRNA in cultured podocytes changed cell morphology from an elongated to a round shape along with a change in ezrin distribution 18 . Normally, podocytes are sequestered from platelets, but when the glomerular barrier is injured, podocytes gain access to CLEC-2 on platelets. In addition, a soluble form of CLEC-2 with molecular weight 25 kDa exists in human plasma 22 . We speculated that CLEC-2, the known ligand for PDPN, may have some biological impact on podocytes. This hypothesis is supported by previous reports that injection of antibodies against specific epitopes of PDPN caused transient proteinuria and foot process effacement in rats 23 . In the present study, we examined the effect of CLEC-2 in podocytes in vitro and in vivo.

Results
Effects of CLEC-2 on in vitro podocytes. We first tested the effects of recombinant Fc-human CLEC-2 on cultured mouse podocytes. Although most proteins characteristic to podocytes, such as nephrin and podocin, are rapidly downregulated upon in vitro culture, PDPN staining was maintained in primary cultured podocytes with similar intensity to in vivo podocytes (Fig. 1A). As reported previously 3 , a pull-down experiment showed that Fc-human CLEC-2 bound to mouse PDPN (S. Fig. 1A). The majority of podocytes treated with Fc protein for 1 h showed an elongated morphology with sharp protrusions and had numerous F-actin filaments. In contrast, podocytes treated with Fc-CLEC-2 showed a round cell shape without sharp protrusions and a decrease in F-Actin (Fig. 1B). Furthermore, podocytes treated with Fc-CLEC-2 showed less adhesion to the collagen-1-coated plate within 1 h than the Fc control cells (59.6% vs. 65.3%) (Fig. 1C). Podocytes with Fc-CLEC-2 showed more migration than the Fc control (2.62 vs. 1.93 mm/24 h) (Fig. 1D).
We next studied the phosphorylation status of ERM, which links PDPN with F-actin, to reveal the intracellular signaling induced by CLEC-2. Western blot analysis revealed that treatment with Fc-CLEC-2 reduced the pERM/ERM ratio in cultured podocytes, indicating that CLEC-2 induced dephosphorylation of ERM proteins (Fig. 1E, S.Figs. 2 and 3). Ezrin has been regarded as the major ERM protein in podocytes 24 . Using Western blot analysis and quantitative PCR, we found that ezrin was downregulated in cultured podocytes (S. Fig. 4A and B). Ezrin was not detected by immunostaining in cultured podocytes. Moesin was intensely stained in the protrusions of the Fc control podocytes, and incubation with Fc-CLEC-2 markedly decreased moesin staining (Fig. 1F, S. Fig. 5). These results collectively indicate that CLEC-2 induced the dephosphorylation of ERM proteins, which caused the dissociation of F-actin filaments from PDPN, F-actin degradation, and cell morphological changes.

The effects of CLEC-2 on in vivo podocytes.
To test the effects of CLEC-2 on in vivo podocytes, we generated a new recombinant CLEC-2 protein with a smaller size, because the above Fc-CLEC-2 forms a tetramer with a size of about 240 kDa, which was not expected to reach podocytes through the normal glomerular barrier. The new mouse CLEC-2 protein, FLAG-CLEC-2, exists as a monomer in solution and the size is 30-35 kDa. FLAG-CLEC-2 bound to cultured podocytes, which was competed with Fc-CLEC-2, but not with Fc, confirming the specificity of the binding (S. Fig. 6).
We infused 5 μg/g body weight of FLAG-CLEC-2 into normal mice and excised the kidney 1 h later. Double immunostaining for FLAG and nephrin, a protein specific to podocytes, confirmed that FLAG-CLEC-2 bound to podocytes ( Fig. 2A). However, when glomeruli were isolated from mice 1 h after injection of FLAG-CLEC-2, FLAG was not detected by Western blot in the glomerular lysate. This implies that FLAG-CLEC-2 was not internalized into podocytes and that FLAG-CLEC-2 was detached from glomeruli during the glomerular isolation procedure. To test this possibility, cultured podocytes were incubated with FLAG-CLEC-2 (10 μg/mL) at 37 °C and washed with an acidic buffer. FLAG staining in podocytes decreased after washing. When incubation was performed at 4 °C, FLAG was stained in podocytes with similar intensity to those incubated at 37 °C, and the   www.nature.com/scientificreports/ staining was similarly decreased after washing with an acidic buffer (Fig. 2B). These observations indicate that FLAG-CLEC-2 is not internalized into podocytes. Quantitative RT-PCR analysis of the glomerular RNA revealed that infusion of FLAG-CLEC-2 increased Serpine1 mRNA, a podocyte injury marker 25 , 2.45 (1.68-8.14)-fold compared to controls (Fig. 2C). The Western blot of glomerular lysate revealed that FLAG-CLEC-2 decreased the pERM/ERM ratio 0.65 (0.38-0.76)-fold (Fig. 2D, S.Figs. 7 and 8), indicating that CLEC-2 induced dephosphorylation of ERM proteins. This was also confirmed by double immunostaining of pERM and podocalyxin (Fig. 2E).
In scanning electron microscopy (SEM) images, widening of foot processes was observed in 18.5% of visual fields in the mice treated with FLAG-CLEC-2, contrasting with no such change in control mice, suggesting that CLEC-2 induced a change in the cytoskeleton in foot processes (Fig. 2F).
These findings indicate that CLEC-2 induced dephosphorylation of ERM and concomitant widening of the foot processes of podocytes. www.nature.com/scientificreports/ Urinary platelets excreted by mice with podocyte injury. We tested the possibility that podocytes can encounter platelets when glomeruli are injured. For this purpose, we induced podocyte injury in NEP25 mice by injecting LMB2. Five days after the injection of LMB2 (5 ng/g body weight), NEP25 mice exhibited both urinary protein and urinary blood (Fig. 3A), indicating disruption of the glomerular barrier. Immunostaining of CD41 revealed that CD41 positive platelets were observed in the urinary sediments at this time point (Fig. 3B), but not in those from LMB2-untreated control mice (S. Fig. 9A). To further verify leakage of platelets, platelets were collected from wild-type mice, labeled with DiI, and injected into NEP25 mice 5 days after the injection of LMB2. DiI-labeled platelets were found in the urine collected from the injected mice (Fig. 3C). Thus, platelets in the bloodstream can make contact with podocytes when the glomerular barrier is injured.

Discussion
The present study revealed that recombinant CLEC-2, the ligand of PDPN, induced significant morphological change, attenuated adhesion, and promoted migration in cultured podocytes. CLEC-2 causes dephosphorylation of ERM and decomposition of PDPN-ERM-F-actin complex. A previous report indicated that PDPN binds to ezrin, whose phosphorylated form tightly connects with F-actin in podocytes 18 . A similar phenomenon has been reported in the FRCs of lymph nodes. CLEC-2 on dendritic cells acts on the PDPN of FRCs and induces dephosphorylation of ERM, disconnection of ERM from the plasma membrane, and a reduction in actomyosin contractility, which elongates FRCs 26 . In both FRCs and podocytes, binding with CLEC-2 attenuates the basal function of PDPN. Similarly, CLEC-2 inhibits the basal function of PDPN in keratinocytes and lymphatic endothelial cells 11,27 , but in these cases the net effect of CLEC-2 is the inhibition of cell migration, which is opposite to that in podocytes. The reason for the opposite direction is not clear, but differences in cell character may be involved. Podocytes are basically static cells and have unique thick actin bundles. In addition, a recent study showed that the stimulation of PDPN acts downstream of VEGF signaling, but not directly on ERM, in lymphatic endothelial cells 28 . Transient exposure to recombinant FLAG-CLEC-2 protein in a short period (1 h) induced a significant morphological change in intact in vivo podocytes although the effect was modest compared to those in cultured podocytes. The modest effect may be caused by the monomeric feature of FLAG-CLEC-2. In injured glomeruli, polymeric CLEC-2 on platelets may bind to PDPN on podocytes and exert greater impacts. Moreover, in injured glomeruli, nephrin and podocalyxin are rapidly and remarkably downregulated 29 , which bind to NHERF2 and stabilize ezrin-F-Actin complex in intact podocytes 20,21 . Therefore, CLEC-2 may have a more significant impact on injured podocytes than those of the perfusion study.
CLEC-2 is a membrane-bound protein mainly expressed in platelets. In addition, CLEC-2 exists in the circulation as shed or microparticle-bound forms 30 . It was reported that the mean plasma concentration of these soluble forms of CLEC-2 was 59-100 pg/mL in healthy volunteers and was increased to 260-380 pg/ml in patients with platelet-activating diseases 22 . In some patients with thrombotic microangiopathy or disseminated intravascular coagulation, the concentration of soluble CLEC-2 exceeds 1000 pg/ml [31][32][33] . Because platelets are retained in inflamed glomerular capillaries 34 , the local concentration of soluble CLEC-2 may be higher in glomerular diseases. Nevertheless, these concentrations do not appear sufficient to induce morphological change in normal podocytes considering the high dose of the recombinant CLEC-2 (5 μg/g body weight) used in the present study.
Platelets are smaller than erythrocytes, therefore they can pass through the damaged glomerular barrier in glomerular diseases with hematuria. In fact, urinary platelets were detected in glomerular diseases [35][36][37][38] . We also demonstrated that platelets are excreted into urine in our podocyte injury mouse model. We speculate that platelets may pass through the glomerular barrier and act on PDPN in podocytes in kidney diseases. Although urinary platelets have received almost no attention, they may reflect a distinct disease condition.
Taken together, we propose that PDPN on podocytes works as a sensor of platelet CLEC-2, which is leaked through glomeruli after severe injury. Stimulation by CLEC-2 induces morphological change and detachment of podocytes, which appears to further aggravate podocyte injury. Considering that PDPN on podocytes and CLEC-2 on platelets are evolutionally conserved, this system may have some beneficial effects, such as facilitating the repair process. Further study is necessary to establish the role of PDPN on podocytes.

Animal ethics. All animal experiments were approved by the Animal Experimentation Committee of Tokai
University School of Medicine. All animal experiments were performed in accordance with relevant guideline and regulations, and the study is reported in accordance with ARRIVE guidelines (https:// arriv eguid elines. org).
StepTagII-3FLAG-CLEC-2, a fusion of StrepTactin FLAG double tags and the C-terminal extracellular domain of mouse CLEC-2 (51-229), was generated in HEK293 cells transiently transfected with the expression plasmid using polyethyleneimine Max reagent (Polysciences, Inc.). The supernatant of cell medium was added with biotin blocking solution (Biolock, iba Life Science), and StepTagII-FLAG-CLEC-2 protein (hereafter designated as FLAG-CLEC-2) was purified by a StrepTactin Sepharose column (StrepTrap HP, GE healthcare Life Sciences). Mass spectrometric analysis by the LCMS-IT-TOF (Shimadzu) confirmed that the purified protein  Fig. 1B). The deglycosylation by PNGase F (New England Biolabs) changed the two bands to a single band (S. Fig. 1C). Pull-down assay confirmed that mouse PDPN can bind FLAG-CLEC-2 (S. Fig. 1B).
Blue Native PAGE showed that Fc and Fc-CLEC-2 exist as tetramers, and FLAG-CLEC-2 exists as a monomer in aqueous solution (S. Fig. 1D).

Western blot analysis.
Cells were lysed in a lysis buffer containing 1% Triton X-100, 2 mM CaCl 2 , 0.5 mM PMSF, protease inhibitor cocktail (cOmplete, Roche), and 50 mM Tris/HCl (pH7.4). For analysis of phosphorylated protein, 10 mM NaF, 1 mM Na 3 VO 4 , and 5 mM Na 4 P 2 O 7 were added. The homogenates were centrifuged at 15,000 rpm to remove the insoluble fraction. Each protein sample was separated by SDS-PAGE and transferred onto a PVDF membrane. The protein-blotted membranes were incubated with 1:1000 diluted primary antibodies overnight at 4 °C and then incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The density of the positive bands was quantified by image analysis with CS Analyzer 3.0 (ATTO). The following primary antibodies were used: pERM (Cell Signaling, #3726), ERM (Cell Signaling, #3142), and β-tubulin (Cell Signaling, #2128).
Pull-down assay. Podocyte lysate (1.5 g/L, 50 µL) containing 1% Triton X-100 was mixed with 3 μg of Fc-CLEC-2 or FLAG-CLEC-2 for 1 h at 4 °C, and the samples were incubated with KANEKA KanCapA (Wako) or Strep-Tactin Superflow plus (Qiagen), respectively, for 1 h at 4 °C. After removing the supernatant, the beads were washed three times with cell lysis buffer and then incubated in SDS sample buffer. The supernatants were subjected to SDS-PAGE followed by Western blot with anti-PDPN antibody.
Isolation of glomeruli and primary podocyte culture. Glomeruli were harvested using the bead method as previously reported 29 . They were cultured on a collagen-1-coated dish for 7 days in DMEM/F12 medium containing 5% FCS and 0.5% ITS-A. Outgrowing cells were detached and passaged after removing residual beads and glomeruli. Cells were used for in vitro experiments 1 to 7 days after the first to third passages. Cultured podocytes were treated with Fc or Fc-CLEC-2 (10 µg/mL).

Isolation of in vivo podocyte mRNA.
We utilized RiboTag mice, which express the ribosomal protein, Rlp22, which is tagged with hemagglutinin (HA) only in cells expressing Cre recombinase 40 . The RiboTag mice were crossed with podocyte-specific Cre-expressing mice (Nphs1-Cre mice). Podocyte RNA was purified from podocyte polysomes obtained by immunoprecipitation with anti-HA antibody of glomerular lysate.
Adhesion assay. Primary cultured podocytes were seeded at a density of 30,000/cm 2 in collagen-1-coated 96-well plates. After 1 of hour incubation at 37 °C with Fc or Fc-CLEC-2 (10 µg/mL) in 0.5% FCS medium, all wells were washed several times with PBS. The number of attached cells was quantified by Cell Counting Kit-8 (Dojindo Laboratories).
Migration assay. Primary cultured podocytes were seeded at a density of 30,000/cm 2 within O-rings on collagen-1-coated dishes and cultured to reach 90-100% confluency. After the O-rings were removed, the cells were allowed to migrate for 24 h at 37 °C in 5% FCS medium with Fc or Fc-CLEC-2 (10 µg/mL). The longest migration length was measured.
Perfusion of mouse kidney with recombinant CLEC-2 protein. C57BL/6 mice (4-6 months of age, approximately 12-19 g body weight) were used for the experiments. Under anesthesia with pentobarbital (50 mg/kg, i.p.) and buprenorphine (0.05 mg/kg, s.c.), the celiac and superior mesenteric arteries were transiently occluded with clips. The kidneys were perfused with 300 μl of PBS or PBS containing 5 μg/g body weight of FLAG-CLEC-2 through a catheter placed in the abdominal aorta at a distal site and then the clips were    www.nature.com/scientificreports/ followed by 4% PFA/PBS for 1 h before preparing frozen blocks with OCT to preserve the phosphorylation of ERM. Information regarding antibodies is shown in Table 1. Can Get Signal solution (TOYOBO) was used for FLAG** and nephrin staining of frozen kidney sections.
Electron microscopy. Kidneys were fixed by perfusion with 4% PFA and then immersed in 2.5% glutaraldehyde for 30 min. Subsequent preparation of SEM was performed using standard methods. We randomly selected 8-10 glomeruli in each mouse and 3 images at X8000 magnification were captured for evaluation of foot processes. The number of images containing foot processes that were more than twice as thick as normal ones were counted.
Urinary platelets in mice with podocyte injury. As a podocyte injury model, we used NEP25 mouse line 41 , which expresses human (h) CD25 selectively in podocytes. Injection of an hCD25-targeted immunotoxin, LMB2, induces podocyte injury dose-dependently. In this study, NEP25 mice were injected with 5 ng/g body weight of LMB2. Five days after the LMB2 injection, urinary protein and blood were detected by Uropaper III UHAGKSpH 7S (Eiken), and then urine was collected and centrifuged at 500 g. The sediment was washed several times with PBS containing 0.1% BSA, EGTA 1 mM and PGE1 0.25 μM and stained with anti-CD41 antibody (Biolegend, #133,9011:100), and then inspected with confocal microscopy (ZEISS, LSM-880).
Injection of DiI-labeled platelet solution into NEP25 mice. Platelets were isolated from the blood of C57BL/6 mice 42 and stained with Vybrant DiI (Thermo Fisher scientific). A separate set of NEP25 mice were injected with LMB2. Five days later, DiI-labeled platelets were injected. The urine was collected and centrifuged at 180 g. Thereafter, the supernatant was centrifuged at 1300 g, according to the platelets isolation protocol 42 . The pellets were washed several times with washing buffer containing BSA, EGTA and PGE1, and then inspected with confocal microscopy (ZEISS, LSM-880).

Statistical analyses.
The results are expressed as the median and interquartile range (IQR). P values of < 0.05 were considered to indicate statistical significance. For Fig. 1B, C and F, differences between groups were analyzed using Paired sampled t-test. For Fig. 1E and 2D, ratio of Fc-CLEC-2/Fc or FLAG-GLEC-2/Control was obtained in each experiment, and the experiments were repeated 4 and 5 times, respectively. A one-sample t test was used to determine whether the mean ratio was different from 1. For other analyses, differences between groups were analyzed using the Mann-Whitney U-test for continuous data. Statistical analyses were performed using the JMP software program (version 11, SAS Institute Inc.; Cary, NC, USA).